DESCRIPTION (taken from the application): The activity of smooth muscle myosin is regulated by phosphorylation of the regulatory light chain (RLC), and proper functioning of smooth muscles requires precise control of the extent of this phosphorylation. This control is achieved by regulating the activities of myosin light chain kinase (MLCK) and the corresponding phosphatase. Whereas MLCK and its regulation by Ca2+-calmodulin is extensively studied, little was known about the corresponding phosphatase until the isolation of a heterotrimeric protein that appears to be the major myofibrillar myosin phosphatase (MP). MP is composed of a targeting, catalytic and small non-catalytic subunits (MPt, MPc and MPs respectively). All three subunits have been cloned. Although reports on the properties of this MP have appeared in the literature, incisive biochemical and structural analyses is hampered by its low natural abundance and purification yield. Our laboratory had succeeded in expressing MPt and the MPc-MPt complex in insect cells, and MPc and MPs in bacteria. In this application we propose to fully characterize the interactions between the MP subunits, and between MPt and myosin using analytical ultracentrifugation, isothermal titration calorimetry, crosslinking and electron microscopy. Specifically, we will 1) Characterize the interactions between MPt and myosin. 2) Identify the sites of interaction between MPc and MPt. 3) Characterize the interactions of MPs and coiled coil regions of regulatory kinases with MPt. 4) Characterize the conformations of MPt and MP. 5) Characterize the effects of phosphorylation on the properties of MPt. Results from these studies will begin to test such hypotheses that MPt functions to position the catalytic site of MPc at the RLC phosphorylation site. They will also provide the groundwork for future structural studies using resonance energy transfer, image reconstruction electron microscopy, X-ray crystallography etc. In the long term, information derived from this project may contribute toward the prevention, diagnosis and treatment of such cardiovascular diseases as hypertension and atherosclerosis.